Gibberellin produced in the cotyledon is required for cell division during tissue reunion in the cortex of cut cucumber and tomato hypocotyls

Abstract

Cucumber (Cucumis sativus) hypocotyls were cut to one-half of their diameter transversely, and morphological and histochemical analyses of the process of tissue reunion in the cortex were performed. Cell division in the cortex commenced 3 d after cutting, and the cortex was nearly fully united within 7 d. 4',6-Diamidino-2-phenylindole staining and 5-bromo-2'-deoxyuridine labeling experiments indicate that nDNA synthesis occurred during this process. In addition, specific accumulation of pectic substances was observed in the cell wall of attached cells in the reunion region of the cortex. Cell division during tissue reunion was strongly inhibited when the cotyledon was removed. This inhibition was reversed by applying gibberellin (GA, 10(-4) M GA3) to the apical tip of the cotyledon-less plant. Supporting this observation, cell division in the cortex was inhibited by treatment of the cotyledon with 10(-4) M uniconazole-P (an inhibitor of GA biosynthesis), and this inhibition was also reversed by simultaneous application of GA. In contrast to the essential role of cotyledon, normal tissue reunion in cut hypocotyls was still observed when the shoot apex was removed. The requirement of GA for tissue reunion in cut hypocotyls was also evident in the GA-deficient gib-1 mutant of tomato (Lycopersicon esculentum). Our results suggest that GA, possibly produced in cotyledons, is essential for cell division in reuniting cortex of cut hypocotyls.

Figure 1

Schematic illustration of how the hypocotyls were cut. The hypocotyls of 7-d-old plants were cut to one-half of their diameter, transversely, 3 cm from the base, using a razor blade (0.1 mm thick). The plants were then grown for an additional 10 d.

Figure 2

Light micrographs (A–E) and schematic illustrations (A'–E') of the tissue reunion process in the cut hypocotyls of cucumber. A and A', Immediately following cutting of the hypocotyl. Black arrows indicate mucus-like substances stained with toluidine blue O. B and B', One day after cutting. White arrows indicate a wall-like structure intensely stained with toluidine blue O. C and C', 3 d after cutting. White arrowheads indicate a layer of cell wall where the confronted cortex cells attach to each other. D and D', 5 d after cutting. Asterisks indicate randomly directed cell division and intrusive cell elongation. E and E', 7 d after cutting. Black and white stars indicate cells in the tissue reunion region and cells in the non-reunion region, respectively. All sections were stained with toluidine blue O. Arrowheads indicate the location of the cut. co, Cortex; vb, vascular bundle. Scale bars indicate 100 μm.

Figure 3

DAPI staining (A–G and I) and BrdU labeling followed by indirect immunofluorescence microscopy with anti-BrdU antibody (H and J). Sections were prepared from cells from the tissue reunion (A–C, G, and H) and non-reunion (D–F, I, and J) regions. A and D, 3 d after cutting the hypocotyl; B, E, G, and I, 5 d after cutting the hypocotyl; C and F, 7 d after cutting the hypocotyl. G and H, Magnified image of the area marked in B. White arrows indicate BrdU-labeled nuclei. I and J, Magnified image of the area marked in E. White arrowheads indicate the position of the cut. White arrowheads indicate the layer of cell wall where the confronted cortex cells attach to each other. Organelle DNA is seen as small dots. Scale bars indicate 50 μm.

Figure 3

DAPI staining (A–G and I) and BrdU labeling followed by indirect immunofluorescence microscopy with anti-BrdU antibody (H and J). Sections were prepared from cells from the tissue reunion (A–C, G, and H) and non-reunion (D–F, I, and J) regions. A and D, 3 d after cutting the hypocotyl; B, E, G, and I, 5 d after cutting the hypocotyl; C and F, 7 d after cutting the hypocotyl. G and H, Magnified image of the area marked in B. White arrows indicate BrdU-labeled nuclei. I and J, Magnified image of the area marked in E. White arrowheads indicate the position of the cut. White arrowheads indicate the layer of cell wall where the confronted cortex cells attach to each other. Organelle DNA is seen as small dots. Scale bars indicate 50 μm.

Figure 4

Transmission electron micrographs of cell walls. Sections prepared from cells in the tissue reunion region (A–C; shown as black stars in Fig. 2E) and cells in the non-reunion region (D–F; shown as an white star in Fig. 2E) were stained with OsO₄ (A and D), double-stained with OsO₄ and RR (B and E), or double-stained with OsO₄ and RR after 1 n NaOH treatment (C and F). All micrographs were taken 7 d after the hypocotyl was cut. CP, Cytoplasm; M, mitochondria; CW, cell wall. Arrows indicate cell wall stained with RR. Scale bars indicate 500 nm.

Figure 5

Effects of phytohormones on cell division during the tissue reunion process in cotyledon-removed plants. The cotyledon was excised from a 7-d-old plant (A), and a lanolin paste containing GA₃ (B), IAA (C), or D.W. (D) was applied to cover the shoot apex and cut surface of the cotyledon. The final concentration of the phytohormones was 10⁻⁴ m. Black arrows indicate cells in the cortex of the cut hypocotyl. Black arrowheads indicate the position of the cut. All sections were stained with toluidine blue O. co, Cortex; vb, vascular bundle. Micrographs were taken 7 d after cutting. Scale bars indicate 100 μm.

Figure 6

Effects of phytohormone inhibitors on cell division during tissue reunion. Uniconazole-P (10⁻⁴ m) plus 0.2% (v/v) Tween 20 was sprayed onto the abaxial surface of the cotyledon of 5-d-old plants once a day (A and B). After 2 d of treatment, uniconazole-P alone (A) or with 2 × 10⁻⁴ m GA₃ (B) or 0.2% (v/v) Tween 20 (C) was sprayed once a day. Lanolin paste containing 10⁻⁴ m TIBA (D) or D.W. (E) was applied above the above the cut on shoots of 7-d-old plants. Black arrows indicate cells in the cortex of the cut hypocotyl. Black arrowheads indicate the position of the cut. All sections were stained with toluidine blue O. co, Cortex; vb, vascular bundle. Micrographs were taken 7 d after the hypocotyl was cut. Scale bars indicate 100 μm.

Figure 7

Tissue reunion in wild-type (A–C) and GA-deficient mutant (gib-1; D and E) tomato. The hypocotyl of each plant was cut to one-half using the same methods as for cucumber hypocotyls and was observed 7 d after cutting the hypocotyl. A, Wild-type plant. B, Cotyledon-removed plant. C, Cotyledon-removed plant with lanolin paste containing 10⁻⁴ m GA₄ applied to cover the shoot apex and cut surface of the cotyledon. D, gib-1. E, gib-1 with microdrop application of 10⁻⁴ m GA₄ to the shoot apex and base of the cotyledon. Black arrowheads indicate the position of the cut. All sections were stained with toluidine blue O. Scale bars indicate 100 μm.